Degenerative disc disease is an epidemic, ultimately resulting in untenable pain and immobility. In advanced cases, spinal fusion is performed where vertebrae are surgically fixed with a mechanical device and an osteogenic material (bone substitute) is bridged between them in an attempt to induce fusion. Of the 600,000 yearly procedures performed in the US, the most common is posterolateral lumbar arthrodesis but the failure rate can reach 25-40% with standard commercial bone substitutes. The reason for failure rests in part with limited biocompatibility of synthetic bone substitutes, inconsistencies with processed cadaveric bone substitutes and in some cases, health complications caused by supraphysiologic doses of bone morphogenic protein. Autologous bone grafts are much more effective, but the approach is associated with donor site morbidity and the volume of available graft is limited. Compromising strategies that employ bone substitutes and bone marrow aspirate (BMA) are becoming common, but efficacy continues to be limited by the bone substitute. The fact that spine related disability is a growing global problem and standard of care interventions have an unacceptable failure rate clearly demonstrates the need for implants that safely and effectively promote bone fusion. The successes and failures of past spinal fusion strategies indicate that a bone substitute that mimics autograft will meet this need. Therefore, the goal of this proposal is to develop a 3D printed biomimetic bone graft substitute (the scaffold) by an innovative combination of stem cell biology, matrix biology, and biomedical engineering. The scaffold will consist of a tough, porous and flexible nanoengineered hydrogel consisting of gelatin methacrylate (gel-MA) coated with extracellular matrix (ECM) purified from osteogenically enhanced human mesenchymal stem cells derived from induced pluripotent stem cells (OEihMSCs). By mimicking the composition of bone matrix, OEihMSC-derived ECM is highly osteogenic. The gel-MA will be further enhanced by addition of novel silicate nanoparticles that impart stiffness and further stimulate osteogenesis. The scaffold will be designed to drive fusion with efficacy equivalent to autograft, but it will be manufactured from a standardized and sustainable source of materials. To achieve this goal, we will: optimize methodology for the generation of various forms of scaffold with a range of gel-MA, nanosilicate and ECM formulations with variations in macroporosity and stiffness (Aim1), optimize attachment, distribution, viability and osteogenesis of cells on the scaffold using in vitro 3D cell culture assays based on rotating wall bioreactor technology (Aim2) and finally, test the optimized scaffold with human BMA in a rodent posterolateral fusion model, incorporating imaging and biomechanical testing approaches (Aim 3). The rationale for this approach is that it has the capacity to satisfy a need for safe and effective autologous bone repair scaffolds for a rapidly growing population. With the current disposition of the FDA favoring autologous and minimally manipulated cytotherapeutic preparations, this strategy is well suited to clinical translation.